Impact of High Altitude Therapy on Type‐2 Immune Responses In

This article isprotected rights by All copyright. reserved. 10.1111/all.13967 doi: lead todifferences version between this andthe ofPleasecite article Version Record. this as been and throughtypesetting, proofreadingwhich thecopyediting, process, pagination AcceptedThis article undergone has for and buthas accepted been not review publication fullpeer Platz,Switzerland and Allergy of Institute Swiss Corresponding author: Munich, Zentrum Helmholtz Germany Switzerland Strasser ArticleSatitsuksanoa Kubena Croxford BoonpiyathadTadech Patients Altitude of High Impact Articletype : Original MRS. MÜBECCEL AKDIS (Orcid ID 0000: GEDR. TAN (Orcid ID : 0000 TADECHDR. BOONPIYATHAD(Orcid ID : 0000 Fax: +41 81 Fax: 81 4100840 +41 +41 Tel.: 81 140 0848 Email: 6 5 4 3 2 1 Chair and Institute Institute Chair and of Enviromental Medicine Drug Discovery, Hochgebirgsklinik Davos,Davos, Christine Medicine,Phramongkutklao of Bangkok,Department Hospital, Thailand Swiss Institute 4 5 , Nonhlanhla Lunjani 5

, , [email protected] Ellen Herve Farine

Kühne AllergyDavos, Center and Education, for Switzerland Research 1,3 D. , Ge Tan

ofAllergy Asthma Research,University and Zurich, of Renner Idorsia

Article: Basic and Translational AllergyImmunology 1,2,3 Cezmi A. Akdis Cezmi A. - 5 0003 , Anita Dreher,

1 , Gertruda, Capova Therapy , 3,4,6

Peter M.A.Peter Groenen Pharmaceuticals - 1,3 0026 Asthma , Cezmi A., Akdis

, David, Mirer - - 8739)

0002 on Type Switzerland

Research, Obere Strasse Obere Research,

- 1,4 -

9228 0001 , Ma, - - - 3,4 4594) 8690 1 2 Immune Responses in Asthma Responses Immune in 2

, Beate RückertBeate , L rtine Clozelrtine , Hans , td 1,3 5 . , - – , Allschwil,, Switzerland

7647) Mübeccel Akdis

UNIKA

- Werner Duchna Werner

5 , Juliane Juliane Schreiber , - T, TU Munich and TU T, 1 , Pattraporn,

22, CH 22, 1 , Daniel S. Daniel 3,4 , Andrew, L. -

7270, Davos 7270, Davos,

Petr may

This article isprotected rights by All copyright. reserved. functions. response, Conclusions: p patients. Serum IL EA asthma patients and correlated with CRTH2 decreased frequencies of treatment altitude suppressive CRTH2 the of function PGD2exogenous CD4 ILC2, EA the asthma phenotypes as treatment. altitude expressing CD4 Results: using flow cytometry cells, eosinophils, Tregs, lymphoidT and innate cells (ILC) in healthy high controls non Methods: asthma of highimpact on altitude the treatment ofpredominance Background: Abstract response ofImmune asthma treatment high in altitude Short title Acceptedwithatients highbaseline levels. Article after - eosinophilic allergic; N .

altitude

The number number eosinophilsThe resting (both of andactivated) and

+ corrected the Twenty

and CD8

A Asthmain reduced treatment high thetype altitude immune2 + sthma present patients immunological withprofiles, distinct treatment. treatment. After 21 days

and CD8and . type endotype.2 The studyaim ofthis was investigate the to . -

six hospitalized asthma (9 hospitalized patients eosinophilic asthma six allergic; EA, The The Improved control allergicin was asthma particularly evident Furthermore, . +

- T cells T response and attenuated showed cells Treg 5 and IL and 5 - frequency

altitude 21 daysfor altitude were increased CRTH2 expression CRTH2increased expression dysregulated and its + E well the ILC2 as frequency of

T cellsT A and and A - 13 13 decreased PGD2 signalingwas viato diminish CRTH2 found

of CRTH2 of

decreased significantlypatients EA in after 8 +

Tregs Tregs non clinic -

eosinophilic non of of therapy altitude which + al al Treg during climate and immunological response in in and immunological response

enrolled partially s

as as

decreased significantlydecreased in

was

- normalized in the study.

allergic; NN) and from peripheral bloodfrom , CRTH2 ,

treatment in asthma

significant -

expressing CRTH2 during + We We Treg cells in in cells Treg ly reduced s assessed assessed

to to with

9 high -

9 a

all

in This article isprotected rights by All copyright. reserved. unstimulated on blood eosinophils eosinophils exacerbations 11) zone membrane often and by response corticosteroid good inhaled therapy to sputum withassociated tissue and eosinophilia, thickening ofthe basement Eosinophilic (5). therapy allergic sensitization 7) 4) by a defined distinct pathophysiological the such as type 2 mechanism, endotype andendotypicphenotypic the characteristics of patient. n andtargeted diagnosis immune Introduction Innate lymphoidcellsILC: Treg cytotoxic T Tc: Th2: 2 T helper PGD2: NN:Non N Eosinophilic allergicEA: asthma CRTH2: Abbreviations Keywords ot ot only depend E AcceptedNon . . Article A: Non A: . common Allergic isthe asthma most asthma phenoty Improvements theblood in and tiss s : Regulatory: Tcells Subtypes asthma Subtypes of defined by the Prostaglandin D2 - - allergicof a intrinsicasthma includes patients or subset with asthma - regulatory open networks Chemoattractantreceptor homolog on cells expressed Th2 eosinophilic noneosinophilic - : Eosinophils,CRTH2,asthmaT cells, phenotype,high eosinophilic allergic asthma is an indicator for is an indicator eosinophil for activation

and

ing

lower on (8, 9) (8, the the - treatments treatments

asthma

health carehealth -

. These . These patients allergic asthma severity thedisease, but of

is defined as

and byupregulated the (1,2) costs

opportunity new for ue . Asthma Asthma managementcan

type eosinophil (6) show . blood blood

Particularly, of inflammationcomplex and of quite variable (12)

eosinophilia cells/µl),(>350 ia associated withare more interleukin ( . It. is

An is asthmaendotype pe

pathway

CD69 with a importantly expressed ata expressed response

n n earlier

expression expression on altitude. IL)

precision precision

be - 5 (7). 5

individualized on

to standard to

onset

the

fewer fewer

low level without (10, (10, (5 (3, -

This article isprotected rights by All copyright. reserved. therapyaltitude has notbeen in described asthma Accepted were reported memoryeffector cells B transitional and plasmablasts dermatitis, on CD4 expression altitude exposure particle and toexposure sunshineD vitamin stimulate photosynthesis allergen stress,inflammatoryless as less loweravoidance, treatment pressure, asthma patients. tightepithelial bronchial barrierjunction asthma leakinessin patients patients. byenhanced PGD2 asthma patients ILnumbers of severe compared in asthma asthma to mild patients (15). 22) eosinophilicin inflammatoryrole allergy response, and Article CD4 peripheral with production ofand IL cytokines type 2 the involved activation in and of migration Th2 capillary production permeabilitymucus and (DP1) 1 receptor plasma in increased increased allergen exposure asthma and PGD2/CRTH2 cells, mast cells, macrophages,monocytes dendriticcells and on variousexpressed types one oftwo represents prostaglandin receptors. (PGD2) functional D2 . I ncreased circulatingsputum IL and Several studies Severalhave altitude demonstrated studies therapythe efficacy of and cells ILC2s Th2 receptor Chemoattractant homolog (CRTH2) cells expressed on Th2 treatment treatment

(24, (24, 25)

in bronchoalveolarin lavage fluid, high altitude

(15) - 5 (29) axis + + CRTH2 . (23)

CD8 . IL

The The hypothesis the effect of high and IL which In allergic patients, PGD2 asthma the production of show . However,. (16)

+ has

CD25 - that wasthat co . (17) 13 + Moreover, increased

. After an After an . allergenthe challenge, concentration of PGD2 T cellsT

ed - + - causes bronchoconstriction,vasodilation, increases in emerged treatment showed treatment expressin 13

. PGD2 PGD2 are partially. effects D2 mediated by prostaglandin T cells is a feature isa cells severe of T feature

+ significantly decreased significantly activation decreased monocyte and

+ T cell in in cell asthmaticT patients are

of cells; cells; of ILC2s ILC2s , detailed detailed

central memory a - stimulated with IL stimulated with significant into g ILC2 and cells g also responsible ILC2 Th2 are for were found eosinophils, Th2 cells, ILC2, cells, CD8eosinophils, Th2 basophils, .

a Concomitant, changes to changes to the immunological landscape after potential - 5 5 and IL - 4, IL 4, and significant frequency of ILfrequency of

source of source cytokines of type 2 and play a (18)

in sputum after sputum in after allergen challenge in - cells its 9α,11β CD4 5 and IL and 5

. CRTH2. on pathophysiologic factor - - 13 - altitude treatment altitude patient subgroups 2, IL

, eosinophils,, + increases in cells, memory B

T cells cells T reductions in memory Treg producing ILC2s allergic (28) remodeli - - 25 and IL 13 - Furthermore PGF2 metabolite is - . In. children with 13 (19) and CCR7 immune -

asthma producingwere ILC2 (13, (13, 14) . Elevated. ng

basophils and ILC2, and basophils - 33 asthma in

in asthma in . The The present . combines combines anti (26, 27) (26,

CRTH2 is increased (20) cells is , increased increased , . The were detected +

Th2 Th2 for in . levels

CRTH2

atopic atopic allergy treat . cells

(21, is

. is

s H ing of of + ,

igh by T

This article isprotected rights by All copyright. reserved. Accepted ZurichKanton consentbefore informed participationthe in prick skin cells/µl > 350 asthma and healthyat control subjects baseline and score the hospital measurement oxide (FeNO2) proper was used inhaled (Table plus corticosteroid agonists beta recruited subjects control Article 21 daystheir chose stay i average stay SwitzerlandHochgebirgsklinik, above Davos, sea level) (1560m averageand 53.3 aged years Methods treatment. the its and on ILCs,functions CD4 to treatmentresponse in study Subjects - agonists for asesse (30)

Medical history,physicalMedical examination, pulmonaryexhaled test, function nitric observationalThis study2 enrolled inhalation technique inh of

ing in the studyS1). All patients (Table with were short treated

. the study.in showed compliance All patients good Lithium heparinLithium .

T test or or test allergen s immunological changes in thekinetics of different asthma phenotypes

of to classify eosinophilia as he patientsreceived questionnaires on asthma control

21 21 days thical (5 males 4 and females) quick C relief asthma symptomsrelief ommittee

in Davosin in n Davos in to receive high altitude receive Davos to treatment. n in highaltitude

- high altitude coagulated blood blood coagulated werefrom p collected - specific IgE for IgE specific allergy for diagnosis. ) were performed

with asthma with asthma and . aled medication. aled medication. .

All low patientsand locations came from altitude

CD8 T cell subsets, Tregs and CD8 cell TregseosinophilsT subsets, and during

was assessed as wellwas as assessed as with who the visitedfirst Davostime for 9 adult inpatients adult 9 S2

atthe Pulmonary Clinic, study.

specific on theCRTH2specific receptor focus ). No systemic corticosteroid treatment corticosteroid systemic ). No

during

and long

The studyThe was

21 21 days the - first first acting inhaled acting inhaled beta

(14 males . with day T All subj otal count otal eosinophil .

Patients Patients had an s medical historymedical their and treatment approved by the by the approved

of atients withatients - acting inhaled acting inhaled test (ACT) test admission to admission , ects gave ects

15 15 females Nine Nine healthy were -

This article isprotected rights by All copyright. reserved. Accepted stimu PGD2 with to on daythe compare suppressive 0 with PGD2 between without and function bead IL and coated Then MA, according (ThermoFisher, USA), the to Waltham, USA). effector cells)cells (T (BecktonNJ,by III Dickinson, Lakes, Franklin FACSAria using (CRTH2 previously lys ofconcentration 500 then and wholeµl of wasblood PGD2 µl with of µMto stimulated 200 a final 2.5 USA) MI, Ann Arbor, from a ofCRTH2 activation expressing cell D T and subsets. ILCs Article see theMethodprocedures, in section this a Kaluza SoftwareCoulter).a For detailed (Beckman ofreagents description and Coulter, (Beckman cells, PBS Jose, San Biosciences USA) CA, (PBS) for light. 780(eBioscience,dye eFluor CA, San Diego, USA) 30 min 4°C, for from protect untilanalyses.temperature First, 500 whole µl blood Suppression assay Treg for cytometryFlow staining is Stimulation of thePGD2 cells with 10 room 10 min at temperature

. Cells . of red surface red cells blood and of

After that After and Treg, T Treg, T effector, . Cells werewith. stained anti then T effector cellsT effector Peripheral cellsPeripheral blood mononuclear CRTH2 were purified stimulation wholeblood Fresh withwas PGD2 was blood into lithium Whole collected tubes heparin and storedat room +

Treg

innate lymphoinnate described were stained lation. stimulation wasThe vehiclePGD2 10% ethanol in as diluted and s , whole w blood ), CRTH2 1

µM for 1 hour. µM 1 wasfor Then, wholeblood with stained viability dye,

- Indianapolis,USA).IN, Data and figures 2

above

were stained in well 96 Ubottom plate and id cells. id extracellularly for extracellularly withmarkers associated eosinophils, for T - CD4

irradiated cells withwereT cultured .

stock

+ as CD25 , Flow cytometrywas using performed a then s

lysed with 500 µl withlysed 500

for 15 15 min attemperaturefor room and washed solution (10 mM) solution (10 mM)

mAbs mAbs surface cell against molecules with kit violet CellTrace cells proliferation + washed CD127 -

CRTH2 rticle’s Supplements. -

(CRTH2 twice for for - Alexa Flour 647 (AF 647) (BD 647(AFAlexa Flour 647)

of 5

with phosphate buffer salinewith buffer phosphate was diluted

days were stained red cell blood lysis soft buffer performed -

manufacturer's instructions. Treg . Cells werestimulated. also K s were analysed - ) andCD4 PGD2 (Caymanchem, anti to asolution µM 200

+ to evaluate the CD4

with viability 0.5µl - CD3/anti + CD25 + CD25 Gallios Gallios

as as

with + - CD127 CD28 CD28 -

with T

This article isprotected rights by All copyright. reserved. EA NNpatients. and to the between asthma groups. N asthma of history pastasthma, of smoking, difference baselinecharacteristicsThe subject arein shown stay thestudy. asthmatic completed Three patients out dropped of the study (N Results significant. .05werethan less considered groups. Tukey’sand comparisons were multiple usedcalculate to differences between the was to test used pair calculate differences Repeated between points. time ANOVA the Chiusing either CA, La Jolla, Software, subject Baseline USA). werecharacteristics data Germany. levels USA).CA, Jose,(ProteinSimple, San Serum Eosinophil Darmstadt,KGaA, Germany). IL Serum Method in section the this Supplements. article’s same PGD2 the dilution.

AcceptedE Article Patients characteristics analysis Statistical of Detection cytokines serumand in supernatant A), A), ed were

Nine eosinophilic allergic asthma (EA), eosinophilicNine (EA), allergic asthma Statistical analysis wasperformedGraphPadPrism using 7.0 (GraphPad IL Serum shorter than 8

Data we

non s (

as gradedas by pulmonary function) in thegender average proportion, age, analyzed using an IV analyzed an using - eosinophilic non - re presented asstandardre presented mean± error (SEM mean 13 levels w - 21 days thehospital in square test or or test 2 square

For a detailed description reagents of and procedures,see ere

- E allergic (NN

measured using (Merck Instrument Erenna the A A had patients duration longer D Immundiagnostik AG,ELISA from total IgE levels, thelung test,theseverityfunction - way ANOVA. Nonparametricway Wilcoxon ANOVA. - 5 levels5

) patients and and ) patients but not

and asthmaco 9

non

were Table

because of because of body index, mass family history of -

eosinophilic allergic asthma allergiceosinophilic asthma

measured Ella on system measured the -

S1. No significantS1.No derived neurotoxin(EDN) 9

healthy controls (HC) healthy controls ntrol ntrol therapy responses s

of of asthma compared were observed ). Bensheim P

values of since theysince analysed - matched matched ,

This article isprotected rights by All copyright. reserved. CRTH2 2A (CD3(Th) N than (Fig.patients EA patients S4D). S4 treatment EA butin after after 21 dayspatients (Fig.CD4 total numbers of S4A). The numbers lymphocytesabsolute of we with the absolute subgroups. IL with patients highbaseline levels (Fig.1C). c IL The B). groups withcompared at and baseline other decreased treatment after (CD11b eosinophils A (CD11b wasbaseline obs assayed then 21 after daysEA therapy of 1A). (Fig. eosinophils ompared to to groups(Fig.ompared other ILSerum 1C). ctivated eosinophilsctivated (CD11b E -

Accepted IL 5, Article High altitude d High altitude B and C

A had significantly more significantly had A more and analysed T here washere no statisticallyactivated change in CRTH2 significant - Next, we determine To T - 5, IL 5,

+ 13 + Fig.S5A). After otal numbersofeosinophils in were thepatientswith significantly decreased absolute

CD16 Th cells (Fig.Th 2A). absoluteof numbers The frequency and CRTH2 total eosinophilsEA in patientsnumber of (Fig. EDN1D). levels correlated + and CD4 ). The absolute CD8 numbers of - Moreov 13 13 and EDN were patients’at levelsbaseline EA sera in higher

after treatment CD4 by cytometry flow compared (Fig.A decrease significant S1A). to -

Siglec8 EDN levelswere + analysed CD25

d erved of thenumber activated in CRTH2 total eosinophilsnumber of EA in and N + ecreased eosinophils circulating and activated CRTH2 ecreased ,

er, serum IL serum er, + CD8 CD16 the - + CRTH2 CCR3 altitude + changes cell subsets T in occurr no

CRTH2 CRTH2 on cellexpression Patientswith EA T subsets. and and - Siglec8 absolute countsabsolute percentage and of

significant changessignificant in were cells Treg observed (Fig. CRTH2expression subsets onTcell

+ + + CRTH2 CD4 CD16 ) cellscompa

- therapy, EA and N 5 5 and IL still were + + CCR3 CD25

- higher in EA patientsthanEA in higher other asthma

Siglec8 + A CD69 ctivation of these markers significantly decreased - 13 in correlated EA patients with the - + CRTH2 CD127 + After 21 After

+ red to healthyred controls (Fig.baseline at + T cells at T cellsat

) in EA patients(Fig.EA ) in 1 - CD69 5 and IL and 5 - - CD69

E (Treg) populations (Fig. The S3). - + A patientsin a showed reduction

daythe serum treatment, mean ) andactivated CRTH2 baseline wereNNbaseline in higher - 13 levels decreased13 in + ) were ing E +

A A patients (Fig.1 eosinophils +

after 21 days therapy,after of

T cells decreased also

CRTH2 in EA NN and higher EA in eosinophils eosinophils B -

aftertreatment eosinophils

and Fig.S1

+ (Fig. S2A and

T - helper helper - + E

+ Treg ).

were

. B). B). s

This article isprotected rights by All copyright. reserved. Accepted any find between correlation circulating ILC2sser and CD127 (Fig.baseline S7at showed also (FigEA in patients 3A B). and The ILC1s ILC3sNCRfrequency and of identifiable relation significantly other in ILC to decreased21 populations after days asthma patients and healthy (CD45 wholeblood in subsets (Fig. The S6). Article frequencies and cellularity CRTH2 of analysisThis a cells showed negative correlationwith IL serum Furthermore of the results CD8 (Fig. withcompared During2C). controls healthy 21 daystreatment, the of (CD3 Tc (Fig. Beforealtitude the 2B). treatment, frequency absolute and numb CRTH2 (Fig. healthythan controls Interestingly,2B). we significantly observed reduced (CD3 High altitude + + CRTH2 CD4 + We We + Lin + CD161 + - Treg cells among cells Treg phenotypes asthma 21 all days treatment after in of high CD8 5 5 and IL - + analysed CD127 CD25 , we,

+ no significantno differenc CRTH demonstrate + T cells showed cells T CRTH2 + c

- r analysed + kit - educed + CD127 13 levels. absolute numbers CRTH2 The frequencyof and CD161 2 - A CRTH2) in NN inpatientstreatmentCRTH2) (Fig. didafter not S7A). We total ILCs

expression on cellexpression on T subsets + -

) cells revealed also increased also in) cellsrevealed EA N and C ). The ILC1s). The frequency of - CRTH2

s CRTH2 expression onCRTH2 treatmentexpression after ILCs CRTH2 how

that 21 days of highreduces treatment altitude controls Notably,controls at baseline. the ILC2sfrequency of

a (

CD45 CRTH2 expression onsubsets cell expression CRTH2 T decrease in EA N and + ) were higher baseline at + ) showed significant difference no between e e between asthmapatientsand healthy controls - + expressing subsets. cell T circulating Lin serum IL - CD127

+ numbers and ofILC2snumbers percentages - CD161 13 levelsEA in 13 patients (Fig.2D). are shownare was

um cytokine levelsum cytokine (Fig.3C). E

A A (Fig. patients 2C). increased (CD45increased + ) and group 1, 2 ILC3 and in EAin and N

in Fig. S5A Fig.in S5A

E A patients patients A correlates correlates ers ers CRTH2 of +

E and NCR and + A A patients - Lin

C. The The + -

with -

This article isprotected rights by All copyright. reserved. Acceptedwhich partially 21stimulation, treatment. after days recovered of CRTH2demonstrated that in significantly higher CRTH2 We also culture stimulated 5A with CRTH2was whenhigher cultured also PGD2(Fig. stimulatedcultures, In 5A). Treg expression to (Fig.compared healthy 4D). controls Article patients asthma cells in wasbaseline PGD2 at in stimulated higher wholeblood 4B days21 CD4 healthy cont indecreased asthmapatients whole stimulated blood,CD69 afte any detect difference significant activatedfrequency of (CD69 cell weT and subsets, whole stimulated blood the withand measured PGD2 Tcell and ILCs Decreased suppressive in function decreasedHigh altitude toPGD2 response inCRTH2 stimulation ). Moreover,CRTH2 - r 21in days in treatment highaltitude patients asthma (Fig. of 4

+ s D andFig.S8B CD25

of We thetreatment determine To of effect

of treatment of analysed asthma patients, which next . + CD127 There wasThere rols (Fig. 4A and Fig. S8A). rols (Fig. Fig. and S8A). Interestingly,4A CD4 activated

ssessed s

the , whereasPGD2 stimulation no effect showed CRTH2 with - CRTH2 - in PGD2

D) supernatant

+ a relatively a weak CRTH2 suppressor of function positive Treg . also the We found frequency of

functional functional + +

Treg , s + and +

+ ) cells. In wholeIn ) cells. unstimulated blood, notwe could after 21 days in Davos stimulated peripheral peripheral stimulated ofasthmablood patients Treg showed suppressive i less function ILC2s as a a ILC2s wereas proportion of significantly on s wasreconstituted partially

CD8 ha s

activated cell numbers between and baseline in the cellsand cultured properties of of properties

d with stimulation (Fig. PGD2 5B). we Thus,

+ defective CRTH2

t he frequency of T effector cell proliferation CRTH2 + on Treg

the +

T cells suppressive functionsuppressive PGD2 after

expressing Treg s Treg

functional compared to CRTH2

, while there was no changewhile wasin, no there s

were strikingly reduced with and without CRTH2 without with and CD69

after 21 days of after 21 days treatment of

found found PGD2 + CD8

n thePGD2 s IL

response ILCs in + - CD25 + – 4 levels4 CRTH2

D - -

Treg expressing expressing ). In PGD2 - CRTH2 s + - - (Fig. to be to

Treg

T (Fig. after +

s , - . This article isprotected rights by All copyright. reserved. CRTH2 patients negativeeosinophilic had with correlation frequency the of CRTH2 between in in observed eosinophilic was weak allergic correlation and a whereas asthma, there ILC2s clinical subsets, and (Fig. responses S9C). A correl strong ofasthma showedphenotype differences (Fig. patients 6C). immuneThe network in analysesof the markers FeNO levelsFurthermore, negative co showeda correlated with thenumber correlation and CRTH2 frequency of CRTH2number of lymphocytes levelsdecreased FeNO and and CRTH2of Furthermore with correlation control significantly increa lymphocytes,numbers of CRTH2 serum IL and a positivelevels correlationwith had of thenumber eosinophils,activated eosinophils significantly lung inand asthmapatients inflammation (Fig. FeNO decrease S9A). responses

Acceptednon Article exhaledoxide correlated and nitric immuAsthma FEV1 control, with

- In theasthma analysis,In withphenotype patients EA N and Overall, eosinophilic noneosinophilic that determined by increased ACT score + + Treg cells (Fig. S10 (Fig. cells Treg

Tc cells cells Tc asthma phenotypes +

with FeNO levels, serum with lymphocytes levels,EDN levels FeNO serum and (Fig.6C).

Tc (Fig. cells N In 6C). , FeNO, levels a negative correlation had - 5 asthma in patients, altitude decreased (Fig. Improved lung correlatedS9B). with function reduced + sed control (Fig.asthma after treatment Improved6A). asthma

Tc c

- significantly treatment improve ells (Fig. NNpatients,In 6C). ACT scoreshad a positive allergic asthma. Heatallergic correlation correlationand asthma. map graphs CRTH2

number and B ). shownare

+ E

Th cells, Th A patients, lungimproved with correlated function and

frequency of correlat

in Fig.in S10A. in correlation and

had ed

eosinophils in EAin a patientsshowed strong rrelation with NN lymphocytesin

negati a

negative CRTH2 + with d

The duration asth of The Th cells. Th

between between eosinophils, T cell asthmalung control, function vely

the the number and frequency (Fig.S9B) +

with the number ofwith number the correlation with the Treg cells (Fig. 6B). Also ation networkwas E A showed different , FEV1 levels, .

ma in ma in non +

Th and Th ne - This article isprotected rights by All copyright. reserved. activation eosinophil byvarious specific after and allergen cytokines specific allergens to 41) i treatment IL regarded induc a and as potent driver as inducedlacking,such sputum and bronchoalveolar lavage low at clinical setting In addition, sampling samples altitude. were bronchial of groupsuch limitations, as another ofasthma comparisonapatients in similar for contribute toto eosinophilic andallergic inflammation. and treatmentin asthma high altitude response immune during dayshigh treatment altitude in 21 of etc. the to related exposome ( triggersairway and asthma exacerbation of inflammation isan stress de and viscosity and concentration promotedthe airthat the of expansionfull ofthe lungs may directaltitude physiological a have thelowerbecause of oxygen benefit therelevantavoidance of allergen duringperiod the wintersummer, of early end to cation eosinophilic reduced hyperresponsiveness,resulting in bronchial blood total eosinophils, and decreased and altitude humidity climates altitude have lowof concentrations reduce corticosteroidsnitric oxide) oral asthma and use in patients life,quality pulmonarydecrease lung of inflammation(decreased function, exhaled Discussion -

AcceptedIL 5, Article . In. patients withnumbers allergic eosinophi asthma, of ) might account changesfor the observed.

pollen are shorter in pollen are duration and lower athighconcentration in altitude FeNO

of asthma pathologyof In eosinophilic andallergic immuneIn type 2 response, asthma, studyThis brings several together novelapproach treatmentAsthma creased lungresistance - 13 , while, n n high and

add

EDN are - on

altitude reduces altitude modif ic protein and HDM

which mayaffect withreduced mediators neuroimmune also

(42) ying . The CD69 expressionThe . of on eosinophils isa marker elevated in in e.g.

(38, 39) (38, high altitude has been showncontrol, has been improvehigh altitude asthma to the immunological characteristics the

pollution, traffic pollution, traffic dietwhilehealthier and hospital, in

er (35)

(32)

simultaneously

of peripheral peripheral sputum and blood eosinophil levels (grass pollen and tree . . Moreover,. moving aw

the CRTH2 is the CRTH2 ofhallmark isthe both and Th2 ILC2 type cytokine2 secretion. . In In to. addition, exposure fungal spores, molds - IgE level eosinophilic

house house dust (HDM) mite increasedto due

(34) therefore controllingtherefore for improved asthma control, FEV1 asthma improved control,

. allergic phenotype

This studyThis thepatients enrolled This study certainThis had )

(36,37) to duringHigh theseason. ay from psychological from ay ls increaseexposure after

de . , while, of effector

tail We show that We .

the the

Other factors (28, 31) (28,

- showing challenge inchallenge is a functional

(40)

cells known the significant . High. .

(33)

A serum that that sthma

, (34, (34, -

This article isprotected rights by All copyright. reserved. t response Accepted treatment chemokin and cytokines CRTH2 except CRTH2controls for and ILC cell subsets CD69 of expression together. Treg patientsallergic with together asthma Interestingly,treatment. and reduced patientstoallergic compared healthy asthma controls after the on CD4 showed CRTH2 expression highly cytokines thanis higher patients healthy controls treatment. the patients Article the in responses inducingin systemic altered treatment high immunologic neutrophilia altitude inflammation.to related neutrophilic conclude we thatThus not could inflammation stimulienvironmental to crucial playappears in a role airway neutrophilia lavagebronchoalveolar of expression higher immunopathologic of nonfeatures lung improved function NN decreased lunginflammation and in Many of thepatients. only CRTH2 activated CRTH2 and asthma patients s . Dysregulation CRTH2. of selective PGD2 activates cells activates PGD2 Th2 ILC2s and CRTH2 decreased peripheral lymphocytesThe blood a showed + CD8

There There are seen are

in o PGD2 -

without eosinophil allergy and

high altitude reducedhigh sensitivity altitude ILC2s CRTH2 and of T c

+ eosinophils comparedEA in patients togroups other but at baseline, .

T In this study,In this wenot measurecytokine did expression expression cellson thebronchoalveolarT in asthma lavage of

hallmark for Th2 cells Th2 for and hallmark

was partial reconstitution cells inducing type2 cytokines and many other pro other many and cytokines type2 inducing cells (43,44) group of . Interestingly,

after allergen 2s (51 RANTES and GM and RANTES have shown to trigger been Th17 +

were no eosinophils decreasedeosinophils therapy. significantly after - were described higher . Our resultsOur . 53) es, which could contribute to eosinophilia eosinophilia to contribute could which es, NN patient + CD8 . W

CRTH2 CRTH2 on expression Treg e demonstrated that t different t + +

Treg CRTH2 challenged T cells.T - allergic asthma are similar to are allergic allergic asthma asthma +

s Th, Th, Treg describe elevated s . dysfunction

- The presentThe that studyof most demonstrated (47, 48)

produced IL CSF receptor in alpha mucosa and in nonin

PGD2 and LTE4 regulate diverse genes in in genes diverse regulate LTE4 and PGD2 antagonists have shown have antagonists between patients asthma healthyand after high 21 days of altitude therapy. ILC2s in human cellsILC2sin (NN) had a(NN) benefit 21 after days had of the and isassociated with (49) - and allergic asthma . In In . . CRTH2 CRTH2 has . been shown to be a

in suppressorin capacit

allergic CD8 , baseline, at (46) -

10 cytokines10 and type 2 -

. Allergen other and + mediated mediated airway CD69

- Tc cells in higher Tc were producing cells producing T are that

asthma, increased type 2asthma, s

was a

expression

(45) correlation

(50) PGD2 efficacy increased increased . Th17 response. Th17 visible (54) - . Our results results Our . inflammatory inflammatory

ell subsets in ell subsets asthma - y of CRTH2 y of activated activated T .

Asthma Asthma

on CRTH2 marker in

with in patients patients in

but +

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This article isprotected rights by All copyright. reserved. diseases. JInvestig Allergol Clin Immunol. 2015;25(1):1 21. 2016;46(6):825 levels of circulating CD4(+) CRTh2(+) T cells characterize 20. chemoattractant receptor preferential induction proinflammatoryof Th2 cytokineproduction through anact 19. SciS A. U 2008;105(50):20009 receptorD2 DPassuppressor a of tumor hyperpermea 18. asthma. Thorax. 2004;59(6):459 PGF2, a PGD2 metabolit 17. patients with mild asymptomatic bronchial asthma. Eur Respir J. 1992;5(2):190 16. Allergy Clin Immunol. 2017;139(4):1379 lipidomic profile and antiviral immunity inresponse to hyaluronan inpatients with severe asthma. J 15. 2017;72(9):1406 molecular profilesbetween children and adults with eosinophilic esophagitis. Allergy. 14. Prostaglandins Oth 13. 2014;44(4):482 12. integrated analysis 11. phenotype 10. polymorphisms affect expression of TH 17 genes. Allergy. 2018;73(6):1342 9. Allergy. 2018;73(2):284 8. neutrophil 7. inadolescent the population. Allergy. 2018;73(8):1744 6. Overview systematicof reviews in allergy epidemiology. Allergy. 2017;72(6):849 5. neutrophilic experimental inasthma mice.Allergy. 2018. and inflammasome 4. forblocks the future precisionof medicine. Allergol Int. 2016;65(3):243 3. Allergy.asthma. 2. Allergy. 2012;67(7):835 1. References

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- 12. k M, Wanke Sokolowska K, BendeljaM, K, Ruckert B, etal.

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56. cells and type 2 innate lymphoid cell

- 60. - altitude treatment: a therapeutic option patients for

cet. 2000;356(9234):982 ay JJ,ay Nathan RA,et al.Asthma Control Test: - li C, MoritaH, Castro 103.

- 82. arge population

- thma:problem a limitedof Berg N, et al.The enen R. Problematic severe - 403. , et al. Treatment for - - - - dependent asthma. 10 e11.

Charvat M, VrijlandtEJ, 7. - Asthma. Am JRespir

90.

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based cohort ult asthmatics. -

5 and5 IL - - altitude induced - - 13 70.

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Accepted Article Hall Fowler IP, AV, Gup PettipherR, Hunter MG, Perkins CM, Collins LP, Lewis BailletT, M, et al.Heightened Bateman ED, Guerreros AG, Brockhaus F,HolzhauerB, Pethe A, Kay RA, al.Fevipiprant,et an G HilveringHinks B, Stoger TSC, L, Marchi Salimi E, ShrimankerM, R, al.Synergisticet Mitthamsiri W, Pradubpongsa Sangasapaviliya P, A,Boonpiyathad T. Decreased CRTH2 Roediger B, Kyle R, Yip KH, Sumaria N, Guy TV, Kim BS,al. et Cutaneous immunosurveillance L,Xue Fergusson J,Salimi M, I,Panse Ussher JE,Hegazy AN, et al. Prostaglandin and D2 Cosmi L, Annunziato F, Galli MIG, Maggi RME,Nagata RomagnaniK, S. CRTH2the is most Robinson HamidDS, Q, Ying S,Tsicopoulos A, Barkans J, Bentley AM, etal. Predomin FajtML, Gelhaus SL, FreemanUvalle B, CE, Trudeau StinsonAmrani SE, BrightlingY, D CE. prostanoid receptor ( 2 Zhao J, Ll PetersSP. Asthma phenotypes: nonallergic(intrinsic) Jasthma. AllergyClin Immunol Pract. Pignatti P, Perfetti L, Galdi PozziE, V,Bossi A, Biale C, Hartnell A,Robinson DS, Kay AB, Wardlaw AJ. CD69 is expressedhuman by eosinophils Pin I,Freitag AP, O'Byrne PM, Girgis HU,Simon Grotzer M, Nikolaizik WH, BlaserK, Schoni MH. High altitude climate therapy - Specific Immunotherapy. Allergy Asthma Immunol Res. 2018; onem S, Berair R, Singapuri A, Hartley R, Laurencin MFM,Bacher G, et al. Fevipiprant,a - dust m - cell - - - 707. 46. 2. - - -

mediated toleranceand contributeto airwayremodeling. Mucosal Immunol. oyd CM, NobleA. Th17 responsesin chronic allergicairway inflammation abrogate inflammatory type 32. -

9. ite allergic asthma. Pediatr Pulmonol. 1994;17(5):304 oids. Eur Respir J. 2017;50(2).

- lymphocytepopulation inatopic asthma. N Engl J Med. 1992;326(5):298 - - blind, parallel 12. ta A, Tetzlaff Nivens K, MC, Sarno M, et al.Efficacy of BI 671800, an

- 2 CD8(+)lymphocytesT by lipid mediators in severe eosinophilic - - 66 e1 stimulation on Innate Lymphoid Cells in Patients With - group, placebo - - Gabardo A, Watson RM, Dolovich J, etal. Changesin the 19. - - 11. 9. -

induced asthmatic responses. Am Rev Respir Dis.

- controlled

2 2 Th and type T2 cytotoxic cells inhealth - JB, Holguin F,etal. Prostaglandin D(2) 44. et al.Increasedet CD69 expression on

chemoattractantreceptor trial. LancetRespir Med. 10(6):662 l/neutrophil crosstalk. J - 11. -

406. - 74.

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- 6. 6):564

- dose antTH2 - - - 73. 304. -

- This article isprotected rights by All copyright. reserved. CRTH2 - ** 0.05, serum between IL and dot) 21 days (blue dot) (NN), =8,and n asthma (HC), healthy compared control =9 n between baseline =9, n non means. as CRTH2 type cells of Expression defined innate2 lymphoid (ILC2s) treatment asthma phenotypes. * serumcorrelation between IL baseline non (EA), =9,non n asthma Th in treatment high altitude. *** < 0.01, and eosinophilseosinophils in activated differe eosinophils. treatment. the compared (HC),=9 n control bet (NA), =9, asthma n non levels ( (CD11b eosinophilsblood ( endotypetype treatment 2 after highaltitude.Absolute in number total peripheral of legend Figure

AcceptedD, Article Figure 4. 4. Figure 3. Figure 2. Figure Figu (CD4 - Frequencyactivated (CD69 of allergic and healthy asthma,n = control 8, (HC),compared =9 n between C re 1. 1. re P + + CD4 CD16 + )

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+ - and CRTH2 z Representative flow cytometry dot ILCs. flowRepresentative plot dot of cytometry CD25 - + C 5 and IL and 5 ation of CRTH2 of ation CRTH2 , Numbers and CRTH2 of Numbers on expression , frequency - 13 with13 and EDNlevels correlated absolute ween baseline P + +

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This article isprotected rights by All copyright. reserved. in responses different asthma phenotyp C, treatment.the (NN),allergic n asthma =9,n non(EA), andexhaled(FEV1) nitricwith patients eosinophilic oxide(FeNO)in allergic asthma ** 0.05, effector assay,Treg: T = (1:1), n proportion ±6. shown Data are means as cytokines IL indicated histogram mean the percentage ( of cells proliferating effectordifferent proportion Treg: conditionsof T numbers and of each the following from * healthy controls. hour PGD2after 1 and stimulation of

Accepted Article Figure 6. 6. Figure A, 5. Figure Heat Heat map vehicle allergic P

5 < 0.01.

days of culturedays with of CRTH2 and ( PGD2 stimulation - 4, IL A, asthma patients (n = 6). Percentage= patients (n6). asthma cells of T effector proliferation Spearman’s correlationresponses between clinical and immunologic

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< 4 This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article